This grant supported the development of a yeast RNA polymerase II transcription system, leading over the past 20 years to the following: isolation of the general transcription factors TFIIB, -D, -E, F, and -H, and the reconstitution of transcription with pure proteins;isolation of the genes for the 16 subunits of the general factors, revealing a remarkable degree of conservation from yeast to humans;discovery of the multiple roles of TFIIH in transcription and DNA repair;discovery of the Mediator of transcriptional regulation;and provision of RNA polymerase II and general transcription factors for X-ray and electron crystal structure determination. The methods developed for these purposes were exploited in the isolation of chromatin remodeling complexes and, most recently, the isolation of genes in their native states. Characterization of the isolated genes revealed the disruption of promoter nucleosomes upon transcriptional activation in vivo. We now wish to reproduce the entire gene activation process in vitro, beginning with a gene in its repressed state, and finishing with transcription. We tried unsuccessfully to establish a gene activation system for the past 20 years. Positive results from a recent breakthrough in the work form a major part of this proposal. We wish to isolate the factors involved and investigate the mechanism of promoter chromatin remodeling. The block to remodeling imposed by heterochromatin will be both amenable to study in the purified system and a source of information about the remodeling mechanism. In parallel with the studies of chromatin remodeling, we will pursue the transcription of naked DNA templates with RNA polymerase II and general transcription factors. Our work of the past several years has overcome longstanding technical limitations and yielded essentially homogeneous, fully assembled pre-initiation complexes. We will introduce purified Mediator and activator proteins to bring about efficient template utilization. Finally, we wish to combine both promoter remodeling and transcription systems to reconstitute the entire gene activation process. Specific aims for the next project period are as follows: 1. Resolution and reconstitution of the newly established PHO5 promoter chromatin remodeling system. 2. Identification of the block(s) to promoter chromatin remodeling of the PHO5 promoter in heterochromatin. 3. Efficient initiation of RNA polymerase II transcription by fully assembled pre-initiation complexes. 4. Coupling of promoter chromatin remodeling and transcription initiation in a complete gene activation system. PUBLIC HEALTH RELEVANCE The significance of the proposed research may be summarized as follows: it will provide the information needed to fully understand the fundamental mechanism of transcription;it will establish a basis for studies of transcriptional regulation;and it will facilitate the design of new therapies for diseases of aberrant gene regulation in the future.